The American Cancer Society recently reported that cancer has overtaken heart disease as the leading cause of death in Americans under the age of 85. Over 1.5 million people will contract cancer in the coming year and close to 570,000 people will die from various cancers. As biotechnology continues to play an ever- increasing role in the clinical treatment and diagnoses of cancer, the development of new clinical biosensors for cancer prognosis is an important and challenging task. Ln vitro selection is a powerful combinatorial technique capable of isolating RNA molecules with a particular binding affinity or catalytic activity from a randomized pool of RNA sequences. High affinity RNA sequences, termed aptamers, have been isolated for a diverse array of targets including proteins and small molecules. Since RNA aptamers display high affinity and high selectivity for their target ligands, they are exciting candidates for development into clinical biosensors. As an example of the diagnostic potential of RNA aptamers, the RNA aptamer specific for theophylline (an asthmatic drug) binds to this drug 10,000-fold better than to caffeine, even though theophylline and caffeine differ by a single methyl group. Our long term goal is to understand how well RNA can function as a tool for high-level molecular discrimination. The primary goal of the proposed research is to develop simple biosensors for cancer prognosis capable of detecting and quantitating urinary levels of pseudouridine and 1-methyladenosine, two important small molecule markers for cancer. The specific aims include: 1. Optimization of a Fluorescent Biosensor Based on the RNA Aptamer for Theophylline. Our current fluorescent biosensor for theophylline will be optimized. Both the limit of detection and selectivity of the biosensor will be assayed. 2. Continued In Vitro Selection of Novel Aptamers for Pseudouridine and 1-Methyladenosine. The novel RNA aptamers will be characterized by biochemical and structural (NMR) studies and will be converted into biosensors using the methods detailed in Specific Aims 1 and 3. 3. A Colorimetric Biosensor for Theophylline and a Direct Fluorescent Biosensor for ATP. Two different approaches for converting RNA aptamers into biosensors will be applied to well- characterized aptamers. [unreadable] [unreadable]